Kidney-urinary tract stone disease (urolithiasis) is a major health problem throughout the world. Most of the stones associated with urolithiasis are composed of calcium oxalate alone or calcium oxalate plus calcium phosphate. Other disease states have also been associated with excess oxalate. These include, vulvodynia, oxalosis associated with end-stage renal disease, cardiac conductance disorders, Crohn""s disease, and other enteric disease states.
Oxalic acid (and/or its salt-oxalate) is found in a wide diversity of foods, and is therefore, a component of many constituents in human and animal diets. Increased oxalate absorption may occur after foods containing elevated amounts of oxalic acid are eaten. Foods such as spinach and rhubarb are well known to contain high amounts of oxalate, but a multitude of other foods and beverages also contain oxalate. Because oxalate is found in such a wide variety of foods, diets that are low in oxalate and which are also palatable are hard to formulate. In addition, compliance with a low oxalate diet is often problematic.
Endogenous oxalate is also produced metabolically by normal tissue enzymes. Oxalate (dietary oxalate that is absorbed as well as oxalate that is produced metabolically) is not further metabolized by tissue enzymes and must therefore be excreted. This excretion occurs mainly via the kidneys. The concentration of oxalate in kidney fluids is critical, with increased oxalate concentrations causing increased risk for the formation of calcium oxalate crystals and thus the subsequent formation of kidney stones.
The risk for formation of kidney stones revolves around a number of factors that are not yet completely understood. Kidney-urinary tract stone disease occurs in as much as 12% of the population in Western countries and about 70% of these stones are composed of calcium oxalate or of calcium oxalate plus calcium phosphate. Some individuals (e.g., patients with intestinal disease such as Crohn""s disease, inflammatory bowel disease, or steatorrhea and also patients that have undergone jejunoileal bypass surgery) absorb more of the oxalate in their diets than do others. For these individuals, the incidence of oxalate urolithiasis increases markedly. The increased disease incidence is due to increased levels of oxalate in kidneys and urine, and this, the most common hyperoxaluric syndrome in man, is known as enteric hyperoxaluria. Oxalate is also a problem in patients with end-stage renal disease and there is recent evidence (Solomons, C. C., M. H. Melmed, S. M. Heitler [1991] xe2x80x9cCalcium citrate for vulvar vestibulitisxe2x80x9d Journal of Reproductive Medicine 36:879-882) that elevated urinary oxalate is also involved in vulvar vestibulitis (vulvodynia).
Bacteria that degrade oxalate have been isolated from human feces (Allison, M. J., H. M. Cook, D. B. Milne, S. Gallagher, R. V. Clayman [1986] xe2x80x9cOxalate degradation by gastrointestinal bacteria from humansxe2x80x9d J. Nutr. 116:455-460). These bacteria were found to be similar to oxalate-degrading bacteria that had been isolated from the intestinal contents of a number of species of animals (Dawson, K. A., M. J. Allison, P. A. Hartman [1980] xe2x80x9cIsolation and some characteristics of anaerobic oxalate-degrading bacteria the rumenxe2x80x9d Appl. Environ. Microbiol. 40:833-839; Allison, M. J., H. M. Cook [1981] xe2x80x9cOxalate degradation by microbes of the large bowel of herbivores: the effect of dietary oxalatexe2x80x9d Science 212:675-676; Daniel, S. L., P. A. Hartman, M. J. Allison [1987] xe2x80x9cMicrobial degradation of oxalate in the gastrointestinal tracts of ratsxe2x80x9d Appl. Environ. Microbiol. 53:1793-1797). These bacteria are different from any previously described organism and have been given both a new species and a new genus name (Allison, M. J., K. A. Dawson, W. R. Mayberry, J. G. Foss [1985] xe2x80x9cOxalabacter formigenes gen. nov., sp. nov.: oxalate-degrading anaerobes that inhabit the gastrointestinal tractxe2x80x9d Arch. Microbiol. 141:1-7).
Not all humans carry populations of O. formigenes in their intestinal tracts (Allison, M. J., S. L. Daniel, N. A. Cornick [1995] xe2x80x9cOxalate-degrading bacteriaxe2x80x9d In Khan, S. R. (ed.), Calcium Oxalate in Biological Systems CRC Press; Doane, L. T., M. Liebman, D. R. Caldwell [1989] xe2x80x9cMicrobial oxalate degradation: effects on oxalate and calcium balance in humansxe2x80x9d Nutrition Research 9:957-964). There are low concentrations or a complete lack of oxalate degrading bacteria in the fecal samples of persons who have had jejunoileal bypass surgery (Allison et al. [1986] xe2x80x9cOxalate degradation by gastrointestinal bacteria from humansxe2x80x9d J. Nutr. 116:455-460). Also, certain humans and animals may maintain colonies of O. formigenes but nevertheless have excess levels of oxalate for reasons which are not clearly understood.
The subject invention pertains to materials and methods which reduce the risk for developing oxalate-related disorders by reducing the amount of oxalate in the intestinal tract. This reduction in the intestinal tract leads to a reduction in systemic oxalate levels thereby promoting good health.
In one embodiment of the subject invention, a reduction in oxalate absorption is achieved by supplying oxalate-degrading bacteria to the intestinal tract. In a preferred embodiment, these bacteria are Oxalobacter formigenes. These bacteria use oxalate as a growth substrate. This utilization reduces the concentration of soluble oxalate in the intestine and, thus, the amount of oxalate available for absorption. A reduction of oxalate in the intestinal tract can also lead to removal of oxalate from the circulatory system.
In a specific embodiment, the subject invention provides materials and procedures for the delivery of O. formigenes to the intestinal tracts of persons who are at increased risk for oxalate-related disease. These bacteria and their progeny replicate in the intestine and remove oxalate from the intestinal tract, thereby reducing the amount of oxalate available for absorption (and/or causing oxalate excretion from the blood into the intestine) and thus reducing the risk for oxalate related disease.
In accordance with the teaching of the subject invention, oxalate-degrading microbes other than O. formigenes which utilize oxalate as a substrate can also be used to achieve therapeutic oxalate degradation thereby reducing the risk of urolithiasis and other oxalate-related disorders. Such other microbes may be, for example, bacteria such as clostridia or pseudomonads.
In one embodiment of the subject invention, the microbes which are used to degrade oxalate naturally produce enzymes which confer upon these microbes the ability to degrade oxalate. In an alternative embodiment, microbes may be transformed with polynucleotide sequences which confer upon the transformed microbes the ability to degrade oxalate. Specifically, the enzymes formylxe2x80x94CoA transferase and oxalylxe2x80x94CoA decarboxylase have been identified as playing a central role in oxalate degradation. In one embodiment of the subject invention, an appropriate host can be transformed with heterologous DNA encoding these enzyme activities thereby conferring upon the transformed host the ability to augment oxalate degradation. The host may be, for example, a microbe which is particularly well adapted for oral administration and/or colonizing the intestines. Alternatively, the host may be a plant which, once transformed, will produce the desired enzyme activities thereby making these activities available in the intestine when the plant material is consumed.
A further embodiment of the subject invention provides plants transformed with oxalate-degrading enzymes wherein these plants have enhanced resistance to fungi which require oxalate for their pathogenesis of plants or which produce oxalic acid as a mechanisms for their pathogenesis of plants.
In a further embodiment of the subject invention, a reduction in oxalate levels is achieved by administering enzymes which act to degrade oxalate. These enzymes may be isolated and purified or they may be administered as a cell lysate. The cell lysate may be, for example, O. formigenes. In a specific embodiment, the enzymes which are administered are formyl-CoA transferase and oxalyl-CoA decarboxylase. In a preferred embodiment, additional factors which improve enzyme activity can be administered. These additional factors may be, for example, oxalyl CoA, MgCl2, and TPP (thiamine diphosphate, an active form of vitamin B1).
Thus, in one embodiment of the subject invention, a reduction in oxalate levels is achieved by administering oxalate-degrading enzymes produced by a recombinant microbe, such as Escherichia coli, which has been transformed to express oxalate-degrading enzymes. The recombinant host may be administered in either a viable or non-viable form. A further aspect of the subject invention pertains to pharmaceutical compositions and/or nutritional supplements for oral administration. These compositions release the oxalate degrading microbes, or oxalate degrading enzymes, in the intestines of humans or animals. Preferably the microorganisms and/or enzymes are encapsulated in a dose delivery system that decreases the probability of release of the materials in the human or animal stomach but increases the probability of release in the intestines. The microorganisms and/or enzymes also may be administered as a constituent of foods, such as milk, meats, and yogurt.
In a further embodiment of the subject invention, a reduction in oxalate absorption is achieved in domesticated, agricultural, or zool-maintained animals deficient in oxalate-degrading bacteria by administering oxalate-degrading microorganisms, plants, and/or enzymes.